Simple, reproducible, scaleable model

Action Points

Note that researchers have created a thymus-like organoid system in vitro that promotes the differentiation of T-cells.

Be aware that, currently, the system uses mouse stromal cells which may induce issues if human compatibility becomes desirable.

A serum-free, artificial thymic organoid (ATO) system with a three-dimensional (3-D) structure is providing a robust new tool for the study of human T-cell differentiation from human hematopoietic stem and progenitor cells (HSPCs), according to researchers.

The artificial thymus or "thymus in a dish" system is a technically simple, reproducible in vitro model that closely recapitulates human thymopoiesis and generates mature, naive antigen-specific T-cells closely resembling T-cells from the thymus and blood, Gay M. Crooks, MBBS, of the David Geffen School of Medicine at the University of California Los Angeles (UCLA), and colleagues reported online in Nature Methods.

Calling ATOs "a new tool for the study and development of stem-cell-based engineered T-cell therapies," Crooks and colleagues pointed out that the in vitro model makes it easy to genetically manipulate hematopoietic and stromal compartments, and produce "naive, unperturbed" antigen-specific T-cells. "ATOs can be used to exploit developmental allelic exclusion of endogenous TCR [T-cell receptor] expression as a novel strategy for generating potentially non-alloreactive antigen-specific T-cells for immunotherapy," the researchers wrote.

"This is a very exciting regenerative tissue project," Crooks said in an online profile, noting that with age, the thymus loses its ability to program T-cells. In older cancer patients, she pointed out, insults such as radiation or chemotherapy "knock out what little thymic function they have left."

Until now, "spatiotemporal complexity of T-cell development in the thymus" has limited the ability of existing in vitro models to fully recapitulate human T-cell development, the researchers explained. Although other labs, including their own, have shown that 3-D organoid systems using mouse or human primary thymic stroma support improved positive selection and maturation of human T cells in vitro, these systems depended on the use of primary thymic tissue, resulting in "high experimental variability."

The new ATO system is "based on a delta-like canonical Notch ligand 1 (DLL1)-expressing stromal cell line and serum-free, off-the-shelf components that support robust differentiation, positive selection and maturation of human CD3+TCR-αβ+ CD8 SP and CD4 SP T cells from CB [human cord blood], BM [bone marrow], and PB [peripheral blood] CD34+ HSPCs," the study authors write. "Mature T-cells that developed in ATOs showed an antigen-naive phenotype, a diverse TCR repertoire, and cytokine production and proliferation in response to antigenic stimuli."

Importantly, the model is scaleable and would be easy for other labs to reproduce and use, the researchers said. The serum-free medium allows the ATOs to remain intact for up to 20 weeks, thus avoiding the frequent transfer of cells onto fresh stromal cells required when using mono-layer systems, they pointed out. "The simplicity of the ATO system permits straightforward adoption of the method in laboratories interested in studying human T-cell development and engineered T-cell therapies."

"This is a very promising technique that could lead to better therapies," Jarrod Dudakov, PhD, of the Fred Hutchinson Cancer Research Center in Seattle, WA, agreed in an interview. The APO system "is a really good model and a really good first step," said Dudakov, who was not involved in the study. "It's incredibly important to create this naive diverse repertoire of T-cells," he noted, adding that the 3-D structure of this in-vitro model plays "a critical role in the positive selection of T-cells."

Potentially, this approach could lead to generation of chimeric antigen receptors (CAR) T-cells, and significantly benefit patients undergoing generally aggressive "conditioning" required prior to hematopoietic stem cell transplantation as well as those with T-cell deficiencies caused by autoimmune disease, severe combined immunodeficiency, and infectious diseases such as HIV, Dudakov confirmed.

"We have to see what happens in patients," he added. "They've gone serum-free, which is great, but they are still using this mouse stromal cell line. There's a bit of a step between that and human studies."

This work was funded by the National Institutes of Health, the Tower Cancer Research Foundation, a UCLA Broad Stem Cell Research Center (BSCRC)

Innovation award, a BSCRC Clinical Fellowship, a Prostate Cancer Foundation Challenge Award and a Jane Coffin Childs Postdoctoral Fellowship. Core services were supported by the UCLA Jonsson Comprehensive Cancer Center shared facility, the UCLA Immunogenetics Center, the UCLA Center for AIDS Research Virology Core Lab and the UCLA AIDS Institute, and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. The study authors disclosed no conflicts of interest.

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